The small regulatory RNAs, microRNAs (miRNAs), play important biological roles in various types of living organisms. In humans, defects in miRNA-dependent gene regulation are implicated in several diseases. Despite these facts, functions of miRNAs in the mammalian skeletal system have barely been studied. The let-7 family miRNAs constitute the largest potion of chondrocytic miRNAs. let-7 miRNAs regulate diverse biological functions that include regulation of developmental timing, cell proliferation and differentiation, cancer metastasis, and stem cell self-renewal. However, the physiological role of let-7 miRNAs in skeletal development is unknown. The goal of this research is to establish the role of let-7 miRNAs during skeletogenesis. We will investigate the role of let-7 miRNAs in the mouse skeletal system by suppressing and overexpressing let-7 miRNAs. In mice, let-7 miRNAs are encoded at 8 genomic loci. This extensive genetic multiplicity makes it difficult to perform a loss-of-function study using the conventional gene targeting strategy. To suppress multiple let-7 miRNAs in a tissue-specific fashion, we will misexpress the RNA-binding protein, Lin28, in the cartilage using a Cre-dependent binary system. Lin28 specifically inhibits processing of primary transcripts of let-7 miRNAs and thereby suppresses let-7 expression. The consequences of Lin28 misexpression in mice will be analyzed. In Aim II, functions of let-7 miRNAs will be investigated by a gain-of-function study. let-7 miRNAs will be overexpressed in the cartilage using the same binary gene expression system. let-7 will also be co-expressed with Lin28 to determine the specific function of Lin28. This project should provide insights into the physiological role of let-7 miRNAs in skeletogenesis. In addition, the Cre-dependent expression systems for Lin28 and let-7 will be useful tools for studying the function of these genes in other tissues. Understanding of individual miRNAs is necessary for the development of potential miRNA-based treatment of skeletal diseases. PUBLIC HEALTH RELEVANCE: The small regulatory RNAs, microRNAs regulate gene expression mainly at the post transcriptional level. let-7 microRNAs are the most abundant miRNA species in skeletal tissues. However, the physiological roles of let-7 miRNAs in skeletal tissues are unknown. This project aims to elucidate let-7 function in the mouse skeletal system using novel genetic tools. Understanding roles of individual miRNAs will provide a basis for potential therapeutic interventions for skeletal diseases.